Vol. 283, Issue 1, 265-273, 1997

Inactivation of Nitric Oxide Synthase by Substituted Aminoguanidines and Aminoisothioureas¹

Donald J. Wolff, Douglas S. Gauld, Matthew J. Neulander and Garry Southan

Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey (D.J.W., D.S.G., M.J.N.), and Intramural Research Support Program, SAIC Frederick, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland (G.S.)

A series of substituted aminoguanidines and amino-substituted isothioureas have been examined as inhibitors of nitric oxide (NO) synthase (NOS) isoforms. Each of the agents produced a time- and concentration-dependent inactivation of the NO-forming activity of the affinity-purified NOS isoforms. These inactivations required exposure of NOS to the drug under conditions that supported catalysis, consistent with the proposal that they act as alternate substrate, mechanism-based inactivators. Of the aminoguanidines examined, 2-ethylaminoguanidine was the most efficient inactivator, exhibiting vs. iNOS an apparent K_I value of 120 µM as measured at 100 µM arginine and a k_inact max value of 0.48 min¹ and thus an apparent second-order rate constant for inactivation of 4.0 mM¹min¹. 2-Ethylaminoguanidine displayed a high isoform selectivity for the iNOS compared with the nNOS and eNOS isoforms. 2-Ethylaminoguanidine inactivated NO synthetic activity in cytokine-induced RAW 264.7 cells as measured at 100 µM extracellular arginine with an apparent K_I value of 55 µM and a k_inact max value of 0.09 min¹. The inactivated RAW 264.7 cell NO synthetic capability was restored over a 3-hr period after drug removal to a value 60% of its pretreatment value. This recovery occurred despite the presence of cycloheximide sufficient to inhibit protein synthesis by >99%. 1-Amino-S-methylisothiourea by contrast with the aminoguanidines was identified as a mechanism-based inactivator selective for the nNOS isoform. In contrast to S-isopropylisothiourea, which was found to be both cell penetrant and reversible, 1-amino-S-methylisothiourea appeared cell impermeable and inhibited NOS enzyme "irreversibly."